Utility of Islet Cell Preparations From Donor Pancreases After Euthanasia.

Patients fulfilling criteria for euthanasia can choose to donate their organs after circulatory death [donors after euthanasia (DCD V)]. This study assesses the outcome of islet cell isolation from DCD V pancreases. A procedure for DCD V procurement provided 13 pancreases preserved in Institut Georges Lopez-1 preservation solution and following acirculatory warm ischemia time under 10 minutes. Islet cell isolation outcomes are compared with those from reference donors after brain death (DBD, n = 234) and a cohort of donors after controlled circulatory death (DCD III, n = 29) procured under the same conditions. Islet cell isolation from DCD V organs resulted in better in vitro outcome than for selected DCD III or reference DBD organs. A 50% higher average beta cell number before and after culture and a higher average beta cell purity (35% vs 24% and 25%) was observed, which led to more frequent selection for our clinical protocol (77% of isolates vs 50%). The functional capacity of a DCD V islet cell preparation was illustrated by its in vivo effect following intraportal transplantation in a type 1 diabetes patient: injection of 2 million beta cells/kg body weight (1,900 IEQ/kg body weight) at 39% insulin purity resulted in an implant with functional beta cell mass that represented 30% of that in non-diabetic controls. In conclusion, this study describes procurement and preservation conditions for donor organs after euthanasia, which allow preparation of cultured islet cells, that more frequently meet criteria for clinical use than those from DBD or DCD III organs.

Function and composition of pancreatic islet cell implants in omentum of type 1 diabetes patients.

Intraportal (IP) islet cell transplants can restore metabolic control in type 1 diabetes patients, but limitations raise the need for establishing a functional beta cell mass (FBM) in a confined extrahepatic site. This study reports on function and composition of omental (OM) implants after placement of islet cell grafts with similar beta cell mass as in our IP-protocol (2-5.106 beta cells/kg body weight) on a scaffold. Four of seven C-peptide-negative recipients achieved low beta cell function (hyperglycemic clamp [HGC] 2-8 percent of controls) until laparoscopy, 2-6 months later, for OM-biopsy and concomitant IP-transplant with similar beta cell dose. This IP-transplant increased HGC-values to 15-40 percent. OM-biopsies reflected the composition of initial grafts, exhibiting varying proportions of endocrine-cell-enriched clusters with more beta than alpha cells and leucocyte pole, non-endocrine cytokeratin-positive clusters surrounded by leucocytes, and scaffold remnants with foreign body reaction. OM-implants on a polyglactin-thrombin-fibrinogen-scaffold presented larger endocrine clusters with infiltrating endothelial cells and corresponded to the higher HGC-values. No activation of cellular immunity to GAD/IA2 was measured post-OM-transplant. Establishment of a metabolically adequate FBM in omentum may require a higher beta cell number in grafts but also elimination of their immunogenic non-endocrine components as well as local conditioning that favors endocrine cell engraftment and function.

Lower beta cell yield from donor pancreases after controlled circulatory death prevented by shortening acirculatory warm ischemia time and by using IGL-1 cold preservation solution.

Organs from donors after controlled circulatory death (DCD III) exhibit a higher risk for graft dysfunction due to an initial period of warm ischemia. This procurement condition can also affect the yield of beta cells in islet isolates from donor pancreases, and hence their use for transplantation. The present study uses data collected and generated by our Beta Cell Bank to compare the number of beta cells in isolates from DCD III (n = 141) with that from donors after brain death (DBD, n = 609), before and after culture, and examines the influence of donor and procurement variables. Beta cell number per DCD III-organ was significantly lower (58 x 106 versus 84 x 106 beta cells per DBD-organ; p < 0.001) but their purity (24% insulin positive cells) and insulin content (17 µg / 106 beta cells in DCD III-organs versus 19 µg / 106 beta cells in DBD-organs) were similar. Beta cell number correlated negatively with duration of acirculatory warm ischemia time above 10 min; for shorter acirculatory warm ischemia time, DCD III-organs did not exhibit a lower beta cell yield (74 x 106 beta cells). Use of Institut Georges Lopez-1 cold preservation solution instead of University of Wisconsin solution or histidine-tryptophan-ketoglutarate also protected against the loss in beta cell yield from DCD III-organs (86 x 106 for IGL-1 versus 54 x 106 and 65 x 106 beta cells respectively, p = 0.042). Multivariate analysis indicates that both limitation of acirculatory warm ischemia time and use of IGL-1 prevent the reduced beta cell yield in islet cell isolates from DCD III-organs.

Prevention of tunneled cuffed catheter dysfunction with prophylactic use of a taurolidine urokinase lock: A randomized double-blind trial.

BACKGROUND: The efficacy and cost-effectiveness of prophylactic thrombolytic locks in hemodialysis patients at high-risk of thrombotic dialysis catheter dysfunction is uncertain. We investigated this question in a double-blinded randomized controlled study. METHODS: Prevalent hemodialysis patients from 8 Belgian hemodialysis units, with ≥2 separate episodes of thrombotic dysfunction of their tunneled cuffed catheter during the 6 months before inclusion, were randomized to either: taurolidine heparin locks thrice weekly (control arm) or the same locks twice a week combined with taurolidine urokinase locks once a week before the longest interval without HD (TaurolockU arm). The primary efficacy outcome was the incidence rate of catheter thrombotic dysfunction requiring thrombolytic locks to restore function. RESULTS: 68 hemodialysis patients (32 controls, 36 urokinase) were followed during 9875 catheter days between May 2015 and June 2017. Incidence rate of thrombotic catheter dysfunction was 4.8 in TaurolockU vs 12.1/1000 catheter days in control group (rate ratio 0.39; 95%CI 0.23-0.64). 15/36 (42%) catheters in the treatment group required at least one therapeutic urokinase lock vs 23/32 (72%) in the control group (P = 0.012). The two groups did not differ significantly in catheter-related bloodstream infection and combined cost of prophylactic and therapeutic catheter locks. The TaurolockU group had a numerically higher number of episodes of refractory thrombosis. CONCLUSIONS: Prophylactic use of urokinase locks is highly effective in reducing the number of thrombotic catheter dysfunctions in catheters with a history of recurring dysfunction. Prophylactic use of urokinase locks did not reduce the overall costs associated with catheter locks and was associated with a numerically higher number of episodes of refractory thrombosis. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02036255.

Formation of amyloid in encapsulated human pancreatic and human stem cell-generated beta cell implants.

Detection of amyloid in intraportal islet implants of type 1 diabetes patients has been proposed as cause in their functional decline. The present study uses cultured adult human islets devoid of amyloid to examine conditions of its formation. After intraportal injection in patients, amyloid deposits <15 µm diameter were identified in 5%-12% of beta cell containing aggregates, 3-76 months posttransplant. Such deposits also formed in glucose-controlling islet implants in the kidney of diabetic mice but not in failing implants. Alginate-encapsulated islets formed amyloid during culture when functional, and in all intraperitoneal implants that corrected diabetes in mice, exhibiting larger sizes than in functioning nonencapsulated implants. After intraperitoneal injection in a patient, retrieved single capsules presented amyloid near living beta cells, whereas no amyloid occurred in clustered capsules with dead cells. Amyloid was also demonstrated in functional human stem cell-generated beta cell implants in subcutaneous devices of mice. Deposits up to 35 µm diameter were localized in beta cell-enriched regions and related to an elevated IAPP over insulin ratio in the newly generated beta cells. Amyloid in device-encapsulated human stem cell-generated beta cell implants marks the formation of a functional beta cell mass but also an imbalance between its activated state and its microenvironment.

Cell Mass Increase Associated with Formation of Glucose-Controlling ß-Cell Mass in Device-Encapsulated Implants of hiPS-Derived Pancreatic Endoderm.

Device-encapsulated human stem cell-derived pancreatic endoderm (PE) can generate functional ß-cell implants in the subcutis of mice, which has led to the start of clinical studies in type 1 diabetes. Assessment of the formed functional ß-cell mass (FBM) and its correlation with in vivo metabolic markers can guide clinical translation. We recently reported ex vivo characteristics of device-encapsulated human embryonic stem cell-derived (hES)-PE implants in mice that had established a metabolically adequate FBM during 50-week follow-up. Cell suspensions from retrieved implants indicated a correlation with the number of formed ß cells and their maturation to a functional state comparable to human pancreatic ß cells. Variability in metabolic outcome was attributed to differences in number of PE-generated ß cells. This variability hinders studies on processes involved in FBM-formation. This study reports modifications that reduce variability. It is undertaken with device-encapsulated human induced pluripotent stem cell-derived-PE subcutaneously implanted in mice. Cell mass of each cell type was determined on intact tissue inside the device to obtain more precise data than following isolation and dispersion. Implants in a preformed pouch generated a glucose-controlling ß-cell mass within 20 weeks in over 60% of recipients versus less than 20% in the absence of a pouch, whether the same or threefold higher cell dose had been inserted. In situ analysis of implants indicated a role for pancreatic progenitor cell expansion and endocrine differentiation in achieving the size of ß- and α-cell mass that correlated with in vivo markers of metabolic control. Stem Cells Translational Medicine 2019;8:1296&1305.

Efficacy of urokinase lock to treat thrombotic dysfunction of tunneled hemodialysis catheters: A retrospective cohort study.

BACKGROUND:: There are few data to inform decisions about the optimal management of occluded tunneled cuffed hemodialysis catheters with thrombolytic locking solutions. The effect of dose, dwell-time, and number of administrations remains controversial. METHODS:: In this retrospective single-center review of tunneled cuffed catheters used between 2010 and 2014, restoration of blood flow as well as pre- and post-pump pressures after either short (30 min) or prolonged (48-72 h) administration of 100,000 IU of urokinase locking solution was evaluated in all thrombotic dysfunctions. We also assessed if multiple urokinase locks for the same thrombotic dysfunction event were more efficient to restore catheter performance than single administration. RESULTS:: Data on 773 thrombotic events in 148 patients (236 catheters) were collected during observation period. After urokinase treatment, blood flow and pre-pump pressure improved (median of 50 mL/min and 20 mmHg) whereas post-pump pressure decreased (median of 15 mmHg) (all P < 0.0001). The short thrombolytic procedure, used in more severely dysfunctional catheters, resulted in significantly larger improvements in catheter function than the long procedure. Multiple administrations for the same thrombotic event further improved access function in case of persisting dysfunction after first lock but had no added beneficial effect if blood flow and/or pump pressures were normalized after first urokinase lock. CONCLUSION:: Both short and prolonged administration of urokinase locks were efficient in restoring blood flow and pre- and post-pump pressures in dialysis catheters with thrombotic dysfunction. Multiple urokinase locks provide added benefit on these outcomes only in case of persisting dysfunction after the first lock.

Increase Functional ß-Cell Mass in Subcutaneous Alginate Capsules With Porcine Prenatal Islet Cells but Loss With Human Adult Islet Cells.

Alginate (Alg)-encapsulated porcine islet cell grafts are developed to overcome limitations of human islet transplantation. They can generate functional implants in animals when prepared from fetal, perinatal, and adult pancreases. Implants have not yet been examined for efficacy to establish sustained, metabolically adequate functional ß-cell mass (FBM) in comparison with human islet cells. This study in immune-compromised mice demonstrates that subcutaneous implants of Alg-encapsulated porcine prenatal islet cells with 4 × 105 ß-cells form, over 10 weeks, a FBM that results in glucose-induced plasma C-peptide >2 ng/mL and metabolic control over the following 10 weeks, with higher efficiency than nonencapsulated, while failing in peritoneum. This intracapsular FBM formation involves ß-cell replication, increasing number fourfold, and maturation toward human adult ß-cells. Subcutaneous Alg-encapsulated human islet cells with similar ß-cell number establish implants with plasma C-peptide >2 ng/mL for the first 10 weeks, with nonencapsulated cells failing; their ß-cells do not replicate but progressively die (>70%), explaining C-peptide decline and insufficient metabolic control. An Alg matrix thus helps establish ß-cell functions in subcutis. It allows formation of sustained metabolically adequate FBM by immature porcine ß-cells with proliferative activity but not by human adult islet cells. These findings define conditions for evaluating its immune-protecting properties.

Heterogeneity in the Beta-Cell Population: a Guided Search Into Its Significance in Pancreas and in Implants.

PURPOSE OF REVIEW: Intercellular differences in function have since long been noticed in the pancreatic beta-cell population. Heterogeneity in cellular glucose responsiveness is considered of physiological and pathological relevance. The present review updates evidence for the physiologic significance of beta-cell heterogeneity in the pancreas. It also briefly discusses what this role would imply for beta-cell implants in diabetes. RECENT FINDINGS: Over the past 3 years, functionally different beta cells have been related to mechanisms that may underlie their heterogeneity in the pancreas, such as the stage in their life cycle and the degree of their clustering to islets with varying vascularization. Markers were identified for detecting these subpopulations in tissues. The existence of a functional heterogeneity in the pancreatic beta-cell population is further supported. Views on its origin and methods for its analysis in pancreas and implants will help guide the search into its significance in beta-cell biology, pathology, and therapy.

Urokinase-containing locking solution in the prevention of dialysis catheter dysfunction: a double blind randomized controlled trial.

INTRODUCTION: The prophylactic use of recombinant tissue plasminogen activator once weekly reduces the incidence rate of tunneled cuffed catheter (TCC) malfunction and bacteremia as compared to the exclusive use of heparin as locking solution. Restricting the use of prophylactic thrombolytic agents to patients with a history of thrombotic TCC malfunction could be more cost effective. We conduct a multicenter, double-blind, randomized controlled trial and test the hypothesis that weekly use of urokinase lock will reduce the incidence of thrombotic malfunction by 50% in prevalent hemodialysis patients with a history of thrombotic malfunction. METHODS: Patients with a history of at least two separate TCC thrombotic dysfunctions treated with urokinase lock during the 6 months preceding inclusion are recruited in eight Belgian dialysis units. Patients are randomized in two groups: the control group receiving Taurolock™-HEP500 (heparin 500 IU/mL, taurolidine, citrate 4%) after each hemodialysis session and the treatment group receiving Taurolock-U 25,000 (urokinase 25,000, taurolidine, citrate 4%) once a week and the standard Taurolock-HEP500 at the end of the two others sessions. The primary outcome is the incidence rate of TCC thrombotic dysfunction defined by the use of urokinase. The secondary outcomes are the incidence rate of TCC removal and systemic thrombolysis. For the study, both patients and healthcare staff are blinded to treatment allocation. CONCLUSIONS: The present trial is the first to investigate the effect of Taurolock-U 25,000 catheter lock once a week as secondary prevention in hemodialysis patients with the highest risk of TCC-related thrombotic dysfunction. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02036255.